达那唑阴道膜剂的研制
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摘要
达那唑(Danazol)，是一合成的17α-乙炔睾酮的衍生物，是一个十分有效的治疗子宫内膜异位的药物，常用的给药方法为口服。但口服的副作用很大，主要为对肝的影响。近来有研究表明阴道给药可克服肝脏首过效应，大大减少用药量减轻毒副反应，所以我们将达那唑制成阴道给药用膜剂。
本研究选用聚乙烯醇(PVA)0486和羟丙 甲基纤维素(HPMC)E50为膜材，采用刮板法制备达那唑阴道膜。
为了提高药物的经粘膜渗透率，我们采用Valia-Chien双室渗透扩散池和高效液相色谱法(HPLC)，以免阴道粘膜进行药膜的体外渗透研究，对促渗剂月桂氮(?)酮(Az)的浓度、主药的浓度、PVA：HPMC的比例二因素进行了考察，结果表明：2.12～14.50％w／w Az对达那唑有促渗作用，Az的渗透促进作用并不随其浓度的增加而增加，而是有一个最佳浓度，本实验中Az浓度为6.24％w／w时可达最佳渗透促进作用，增渗倍数为无Az时的1.7倍。膜片中达那唑浓度对经粘膜渗透速率也有影响，当主药浓度由3.46％w／w提高到17.39％w／w时，经粘膜渗透速率由3.59±0.13μg·cm~(-2)·h~(-1)提高到4.88±0.16μg·cm~(-2)·h~(-1)而当主药浓度提高到27.87％w／w时，经粘膜渗透速率不再增加，选用不同的成膜材料，药物经兔阴道粘膜渗透速率没有显著性差异(P＞0.05)。
本研究还采用溶出仪和HPLC，进行了药膜的体外释放实验。首先对释放介质和转速进行了选择，采用pH=5的40％v／v聚乙二醇400 20％v／v异丙醇磷酸缓冲液和转速为50转／分时能达到较好的质控目的。采用此介质和转速对
小同的膜材和达那哇浓度的仆外释放进行了研究，结果表明释放规律符合
HtalChi方程人。PVA作膜材时的药物释放速率u显低于PVA：HPMC（2山和
卜VA：HPMC（l：l）的药膜（P、0．of）Jfi中达那陛浓度在 8．70～27．87OW／w之间变
化时，体外释放迎率随浓度的增加而增加。
根据仆夕实马结果，选g达刀咄 3．！2 11飞g·C，11一；AZ·121＊g’Clll二 PW
4二71n2·CII飞；HPMC4．37lllg·CI。一；汁油4．％1fig·CIn二；作人杉试H划v处
方，进一步在家兔上进行体内试验的研究。问时与日服达那喳进行比较，口
＊给药剂量门mg叶合’，阴道给药剂量31。侣·咤‘，实验表明阴道给药与4
倍量口服给药相比，阴道给药子亡中药浓比口叩给药的高，而l清浓度比厂1
服给药的低。可预期用于临床时，与日服相比，有相同的疗效而副作用要少。
Danazol,a synthetic derivative of 17 a -ethinyltestosterone,is effective in the treatment of endometriosis. Danazol is widely used as an oral remedy,which causes many side effects,mainly results liver dysfunction. Recently,some studies suggest that vaginal administration can serve as an alternative route for danazol therapy that might produce fewer and less severe side effects. Therefore,we prepared a vaginal pellicle of danozol.
In this study,polyvinyl alcohol (PVA)0486 and hydroxy-propyl methyl cellulose (HPMC) E50 were considered for administering danazol and prepared using a general casting method.
In an effort to enhance the permeation rate of danazol,three methods including the use of penetration enhancers the increase of the drug load of the system,the use of various polyvinyl alcohol,have been studied. The in vitro rabbit vaginal mucosa permeation tests from each pellicle were carried out by Valia-Chien diffusion cell consisting of two compartments and high performance liquid chromatography (HPLC) method. The results showed that the permeation rate of danazol was enhanced by Az. The permeation effect of Az didn't increased with increase of Az concentration. In this study,the best concentration is 6.24%w/v,which caused the best permeation effect,with a 1.7 fold enhancement.
Danazol concentration in the pellicles has also found to have an effect on the permeation. The rabbii vaginal mucosa permeation rate was increased from 3.59+0.13 u g cm-2 h-1 to 4.88 + 0.16 u g cm-2 h-1 as danazol concentration from 3.46%w/v to 17.39%w/w and did not change as the increase of concentration from 17.37%w/w to 27.87%w/w. Various pellicles of PVA:HPMC were found to provide similar rabbit vaginal mucosa permeation (P>0.05).
The in vitro release tests from each pellicle were evaluated using dissolution
medium and stirring rate. It was found that quality control could achieve when using phosphate buffer of 40 %v/v polyethylene glycol 400 with 20%v/v isopropyl alcohol (pH=5) as dissolution medium and 50rmp stirring rate. Using this dissolution medium and this stirring rate,the in vitro release tests various PVA:HPMC pellicles and various danazol concentrations were studied. The results showed that all release profiles followed Higuchi equation. The release rate using PVA as ingredient of pellicle was obvious lower than that of using PVA.-HPMC (2:1) and PVA:HPMC(1:1). The release rate of danazol was affected by drug concentration in PVA:HPMC (1:1) pellicle. The release rate increased as the increased of concentration from 8.70%w/w,17.39%w/w to 27.87%w/w.
According to research results above,danazol vaginal pellicle composed of danazol 3.12mg cm-2,Az 1.12 mg cm-2,PVA0486 4.37 mg cm-2;HPMC4.37 mg cm-2 and glycerol 4.96 mg cm-2 was used in in vivo rabbit studies,which compare with orally. The concentrations in plasma and uterus of danazol in rabbit administrated vaginally or orally have been determined by HPLC methods. The oral dosage was 12 mg kg-1,and the vaginal dosage was 3 mg kg" . The results showed that the concentration of serum treated vaginally were much lower than those of 4-fold danazol amount treated orally,but the uterine tissue concentrations of danazol treated former were higher than those of the latter. To be compared with oral administration,it is expectable that the pellicles administrated through vagina have equal theraputic effects but lower side effects.
本研究选用聚乙烯醇(PVA)0486和羟丙 甲基纤维素(HPMC)E50为膜材，采用刮板法制备达那唑阴道膜。
为了提高药物的经粘膜渗透率，我们采用Valia-Chien双室渗透扩散池和高效液相色谱法(HPLC)，以免阴道粘膜进行药膜的体外渗透研究，对促渗剂月桂氮(?)酮(Az)的浓度、主药的浓度、PVA：HPMC的比例二因素进行了考察，结果表明：2.12～14.50％w／w Az对达那唑有促渗作用，Az的渗透促进作用并不随其浓度的增加而增加，而是有一个最佳浓度，本实验中Az浓度为6.24％w／w时可达最佳渗透促进作用，增渗倍数为无Az时的1.7倍。膜片中达那唑浓度对经粘膜渗透速率也有影响，当主药浓度由3.46％w／w提高到17.39％w／w时，经粘膜渗透速率由3.59±0.13μg·cm~(-2)·h~(-1)提高到4.88±0.16μg·cm~(-2)·h~(-1)而当主药浓度提高到27.87％w／w时，经粘膜渗透速率不再增加，选用不同的成膜材料，药物经兔阴道粘膜渗透速率没有显著性差异(P＞0.05)。
本研究还采用溶出仪和HPLC，进行了药膜的体外释放实验。首先对释放介质和转速进行了选择，采用pH=5的40％v／v聚乙二醇400 20％v／v异丙醇磷酸缓冲液和转速为50转／分时能达到较好的质控目的。采用此介质和转速对
小同的膜材和达那哇浓度的仆外释放进行了研究，结果表明释放规律符合
HtalChi方程人。PVA作膜材时的药物释放速率u显低于PVA：HPMC（2山和
卜VA：HPMC（l：l）的药膜（P、0．of）Jfi中达那陛浓度在 8．70～27．87OW／w之间变
化时，体外释放迎率随浓度的增加而增加。
根据仆夕实马结果，选g达刀咄 3．！2 11飞g·C，11一；AZ·121＊g’Clll二 PW
4二71n2·CII飞；HPMC4．37lllg·CI。一；汁油4．％1fig·CIn二；作人杉试H划v处
方，进一步在家兔上进行体内试验的研究。问时与日服达那喳进行比较，口
＊给药剂量门mg叶合’，阴道给药剂量31。侣·咤‘，实验表明阴道给药与4
倍量口服给药相比，阴道给药子亡中药浓比口叩给药的高，而l清浓度比厂1
服给药的低。可预期用于临床时，与日服相比，有相同的疗效而副作用要少。
Danazol,a synthetic derivative of 17 a -ethinyltestosterone,is effective in the treatment of endometriosis. Danazol is widely used as an oral remedy,which causes many side effects,mainly results liver dysfunction. Recently,some studies suggest that vaginal administration can serve as an alternative route for danazol therapy that might produce fewer and less severe side effects. Therefore,we prepared a vaginal pellicle of danozol.
In this study,polyvinyl alcohol (PVA)0486 and hydroxy-propyl methyl cellulose (HPMC) E50 were considered for administering danazol and prepared using a general casting method.
In an effort to enhance the permeation rate of danazol,three methods including the use of penetration enhancers the increase of the drug load of the system,the use of various polyvinyl alcohol,have been studied. The in vitro rabbit vaginal mucosa permeation tests from each pellicle were carried out by Valia-Chien diffusion cell consisting of two compartments and high performance liquid chromatography (HPLC) method. The results showed that the permeation rate of danazol was enhanced by Az. The permeation effect of Az didn't increased with increase of Az concentration. In this study,the best concentration is 6.24%w/v,which caused the best permeation effect,with a 1.7 fold enhancement.
Danazol concentration in the pellicles has also found to have an effect on the permeation. The rabbii vaginal mucosa permeation rate was increased from 3.59+0.13 u g cm-2 h-1 to 4.88 + 0.16 u g cm-2 h-1 as danazol concentration from 3.46%w/v to 17.39%w/w and did not change as the increase of concentration from 17.37%w/w to 27.87%w/w. Various pellicles of PVA:HPMC were found to provide similar rabbit vaginal mucosa permeation (P>0.05).
The in vitro release tests from each pellicle were evaluated using dissolution
medium and stirring rate. It was found that quality control could achieve when using phosphate buffer of 40 %v/v polyethylene glycol 400 with 20%v/v isopropyl alcohol (pH=5) as dissolution medium and 50rmp stirring rate. Using this dissolution medium and this stirring rate,the in vitro release tests various PVA:HPMC pellicles and various danazol concentrations were studied. The results showed that all release profiles followed Higuchi equation. The release rate using PVA as ingredient of pellicle was obvious lower than that of using PVA.-HPMC (2:1) and PVA:HPMC(1:1). The release rate of danazol was affected by drug concentration in PVA:HPMC (1:1) pellicle. The release rate increased as the increased of concentration from 8.70%w/w,17.39%w/w to 27.87%w/w.
According to research results above,danazol vaginal pellicle composed of danazol 3.12mg cm-2,Az 1.12 mg cm-2,PVA0486 4.37 mg cm-2;HPMC4.37 mg cm-2 and glycerol 4.96 mg cm-2 was used in in vivo rabbit studies,which compare with orally. The concentrations in plasma and uterus of danazol in rabbit administrated vaginally or orally have been determined by HPLC methods. The oral dosage was 12 mg kg-1,and the vaginal dosage was 3 mg kg" . The results showed that the concentration of serum treated vaginally were much lower than those of 4-fold danazol amount treated orally,but the uterine tissue concentrations of danazol treated former were higher than those of the latter. To be compared with oral administration,it is expectable that the pellicles administrated through vagina have equal theraputic effects but lower side effects.
引文
1．卫生部继续医学教育委员会编。妇产科学分册。长春出版社，1999年，p239
2. Janne O, Kauppila A, Kokko E, et al. Estrogen and progestin receptors in endometriosis lesions: Comparison with endometrial tissue. Am J Obstet Gynecol, 1981,141(5):562
3. Musich JR, Behrman SJ and Monon KMJ. Estrigenic and antiestrogentic effects of danazol administration in antiestrogentic effects of danazol administration in studies of estradiol receptor binding. Am J Obstet Gynecol, 1981,140(1):62
4. Pearson K. Danazol and live damage. Lancet, 1980;1:645
5. Kase NG, et al. Principles and practice of clinical gynecology. 2 nd ed. New York: Churchill Livingston Inc, 1990,459-464
6．朱关珍、杜明昆、郑惠娥，等。丹那唑治疗子宫内膜位症的临床研究。中国实用妇科与产科杂志，1995；11(4)：225-226
7. Igarashi M. A new therapy for pelvic endometriosis and uterine adenomyosis: local effect of vaginal and intrauterine danazol application. Asia Oceania J Obstet Gynaecol, 1990; 10(1): 1-2
8. Igarashi M, Iiauka M, Abe Y, et al. Novel vaginal danazol ring therapy for pelvic endometriosis, in particular deeply infiltrating endometriosis. Hum Reprod, 1998;13(7):1952-1956
9．金毓翠、王迪钧、曹霖，等。治疗妇科疾病用局部给药缓释装置，CN2416900Y，2001-1-31
10. Igarashi M. Topical drug delivery systems containing danazol. European patent, 0330786A1
11. Takahiro M, Akitoshi W, Shigeru N, et al. Danazol concentrations in ovary, uterus, and serum and their effect on the hypothalamic-pituitary-ovarian axis during vaginal administration of a danazol suppository. Fertil Steril, 1995,
63(63);1184-1188
12．栗原、秀晴、桥本。坐剂。JP平3-90029，1989-9-1
13．莫英杰、瞿玉文、伍锐铎。醋酸洗必泰药膜的研制。天津药学，1999；11(4)：24
14．奚念朱主编。药剂学，第三版，人民卫生出版社。1994，p396-398
15．杨鹏晖，袁和亮，史晖。米非司酮阴道贴膜的研究。中国医药工业杂志，199425(5)：204
16. Chien YW. Novel drug delivery systems, 2 nd ed, New York: Marcel Dekker, 1992, P541
17．陆彬主编。药物新剂型与新技术。人民卫生出版社，1998年p365
18．中国药典，2000版，二部附录XD第三法，附录77。
19. Digenis GA, Nosek D, Mohammadi F, et al. Novel vaginal controlled-divery systems in corporating. Pharm Dev Technol, 1999; 4(3):421-430
20．王黎明，褚云鸿。达那唑经小鼠阴道和口服给药后血药浓度及子宫内含量的比较。生殖与避孕，1994，14(4)：275-279
21．何怀冰、孙时良、刘德林，等。HPLC-MaxPLOT UV检测法测定血清中丹那唑及其代谢物。药学学报，1988；23(9)：698-702
22．章元沛主编。药理学实验，第二版。人民卫生出版社，1996年，p238
2. Janne O, Kauppila A, Kokko E, et al. Estrogen and progestin receptors in endometriosis lesions: Comparison with endometrial tissue. Am J Obstet Gynecol, 1981,141(5):562
3. Musich JR, Behrman SJ and Monon KMJ. Estrigenic and antiestrogentic effects of danazol administration in antiestrogentic effects of danazol administration in studies of estradiol receptor binding. Am J Obstet Gynecol, 1981,140(1):62
4. Pearson K. Danazol and live damage. Lancet, 1980;1:645
5. Kase NG, et al. Principles and practice of clinical gynecology. 2 nd ed. New York: Churchill Livingston Inc, 1990,459-464
6．朱关珍、杜明昆、郑惠娥，等。丹那唑治疗子宫内膜位症的临床研究。中国实用妇科与产科杂志，1995；11(4)：225-226
7. Igarashi M. A new therapy for pelvic endometriosis and uterine adenomyosis: local effect of vaginal and intrauterine danazol application. Asia Oceania J Obstet Gynaecol, 1990; 10(1): 1-2
8. Igarashi M, Iiauka M, Abe Y, et al. Novel vaginal danazol ring therapy for pelvic endometriosis, in particular deeply infiltrating endometriosis. Hum Reprod, 1998;13(7):1952-1956
9．金毓翠、王迪钧、曹霖，等。治疗妇科疾病用局部给药缓释装置，CN2416900Y，2001-1-31
10. Igarashi M. Topical drug delivery systems containing danazol. European patent, 0330786A1
11. Takahiro M, Akitoshi W, Shigeru N, et al. Danazol concentrations in ovary, uterus, and serum and their effect on the hypothalamic-pituitary-ovarian axis during vaginal administration of a danazol suppository. Fertil Steril, 1995,
63(63);1184-1188
12．栗原、秀晴、桥本。坐剂。JP平3-90029，1989-9-1
13．莫英杰、瞿玉文、伍锐铎。醋酸洗必泰药膜的研制。天津药学，1999；11(4)：24
14．奚念朱主编。药剂学，第三版，人民卫生出版社。1994，p396-398
15．杨鹏晖，袁和亮，史晖。米非司酮阴道贴膜的研究。中国医药工业杂志，199425(5)：204
16. Chien YW. Novel drug delivery systems, 2 nd ed, New York: Marcel Dekker, 1992, P541
17．陆彬主编。药物新剂型与新技术。人民卫生出版社，1998年p365
18．中国药典，2000版，二部附录XD第三法，附录77。
19. Digenis GA, Nosek D, Mohammadi F, et al. Novel vaginal controlled-divery systems in corporating. Pharm Dev Technol, 1999; 4(3):421-430
20．王黎明，褚云鸿。达那唑经小鼠阴道和口服给药后血药浓度及子宫内含量的比较。生殖与避孕，1994，14(4)：275-279
21．何怀冰、孙时良、刘德林，等。HPLC-MaxPLOT UV检测法测定血清中丹那唑及其代谢物。药学学报，1988；23(9)：698-702
22．章元沛主编。药理学实验，第二版。人民卫生出版社，1996年，p238